transfer overnight? - (Aug/02/2005 )
I am trying to transfer a 120 KD protein to nitrocellulose membrane with invitrogen system. their recommended transfer condition is 30v for one hour at room temperature. I found the protein was not well transfered with this condition.
My transfer buffer contains 10% methanol.
Plus, since I am detecting the phosphorylated protein, should tranfer at lower temperature (like 4 degree)? Do I need to transfer overnight for my protein?
I really appreciate your response to this question!
Transfering at 4 degrees (or at least with some means to keep the buffer cool) is always a good idea, as the transfer buffer can get pretty hot.
That being said, 30v sounds very low for a transfer. As does the time frame. I generally tranfer using ~0.4 amps for up to one hour. The power supply I use shows about 150V at 0.4A, so that might give you a better guage of how many volts you can use for transfers. Depending on the size of the protein I will change the transfer duration, but never the amps used.
Thanks for the kind reply!
It seems that you are using a much much higher voltage for transferring the protein. Are you dealing with the similar size of protein? And, are you using the same invitrogen system?
I have used the Bio-rad system for running and transferring gel. I used 81v constant for the same protein, tranferred for 2 hours with 20% methonal. At around 0 degree, it gives a satisfied transfer.
For the invitrogen system, I was told by the specialist from the company that it is not appropriate to use voltage higher than 30-40v.
I have read in other forum that somebody tried to transfer with the invitrogen system at 33v overnight at RT. But at 20 hours, he found the protein was overtransferred (broke thourgh the membrane). I wonder if at 4 degree that would not happen.
I have read in other forum that somebody tried to transfer with the invitrogen system at 33v overnight at RT. But at 20 hours, he found the protein was overtransferred (broke thourgh the membrane). I wonder if at 4 degree that would not happen.
I haven't used the Invitrogen system you describe, nor am I familiar with it. But in response to your last statement, I believe that the reasoning behind preventing excessive heat buildup is not to control the rate of transfer but rather to maintain the integrity of the proteins being transferred. Good luck troubleshooting
hi
voltage depends on the system you use. I use currently semi dry device, and exceeding 25v would dammage the eectrode..
I've tried less voltages. but not for overnight. But my colleague did a transfert yesterday at 5V for more than 4 hours and finished by 1h at 10V. (respectively 1/3 and 2/3 of the voltage we use routinely). Everything was ok. So assuming your do transferts at 30 v i would recommend you to do 5V overnight and your protein won't go through your membrane.
make a dummy assay to check conditions.
I use the wet system and I have transfered a 185 kDa protein with 1 hr transfer at RT at 100 V. It works like a charm. Touch wood.
Also fred had earlier said something about reducign the methanol content of the transfer buffer to 5% to increase protein trnasfer
the post Pria mentionned is available here